A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy

Article9 June 2021Open Access Transparent process A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy Rony Chidiac orcid.org/0000-0002-3642-0651 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada These authors contributed equally to this work Search for more papers by this author Md. Abedin Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA These authors contributed equally to this work Search for more papers by this author Graham Macleod orcid.org/0000-0001-6401-9307 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Search for more papers by this author Andy Yang Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Search for more papers by this author Pierre E Thibeault Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Search for more papers by this author Levi L Blazer AntlerA Therapeutics, Foster City, CA, USA Search for more papers by this author Jarrett J Adams AntlerA Therapeutics, Foster City, CA, USA Search for more papers by this author Lingling Zhang Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Heidi Roehrich Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Ha-Neul Jo Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Somasekar Seshagiri AntlerA Therapeutics, Foster City, CA, USA Search for more papers by this author Sachdev S Sidhu Corresponding Author [email protected] orcid.org/0000-0001-7755-5918 AntlerA Therapeutics, Foster City, CA, USA Donnelly Centre, University of Toronto, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Harald J Junge Corresponding Author [email protected] orcid.org/0000-0003-2458-6010 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Stephane Angers Corresponding Author [email protected] orcid.org/0000-0001-7241-9044 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada AntlerA Therapeutics, Foster City, CA, USA Department of Biochemistry, University of Toronto, Toronto, ON, Canada Search for more papers by this author Rony Chidiac orcid.org/0000-0002-3642-0651 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada These authors contributed equally to this work Search for more papers by this author Md. Abedin Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA These authors contributed equally to this work Search for more papers by this author Graham Macleod orcid.org/0000-0001-6401-9307 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Search for more papers by this author Andy Yang Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Search for more papers by this author Pierre E Thibeault Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Search for more papers by this author Levi L Blazer AntlerA Therapeutics, Foster City, CA, USA Search for more papers by this author Jarrett J Adams AntlerA Therapeutics, Foster City, CA, USA Search for more papers by this author Lingling Zhang Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Heidi Roehrich Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Ha-Neul Jo Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Somasekar Seshagiri AntlerA Therapeutics, Foster City, CA, USA Search for more papers by this author Sachdev S Sidhu Corresponding Author [email protected] orcid.org/0000-0001-7755-5918 AntlerA Therapeutics, Foster City, CA, USA Donnelly Centre, University of Toronto, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Harald J Junge Corresponding Author [email protected] orcid.org/0000-0003-2458-6010 Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Stephane Angers Corresponding Author [email protected] orcid.org/0000-0001-7241-9044 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada AntlerA Therapeutics, Foster City, CA, USA Department of Biochemistry, University of Toronto, Toronto, ON, Canada Search for more papers by this author Author Information Rony Chidiac1, Md. Abedin2, Graham Macleod1, Andy Yang1, Pierre E Thibeault1, Levi L Blazer3, Jarrett J Adams3, Lingling Zhang2, Heidi Roehrich2, Ha-Neul Jo2, Somasekar Seshagiri3, Sachdev S Sidhu *,3,4,5, Harald J Junge *,2 and Stephane Angers *,1,3,6 1Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada 2Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA 3AntlerA Therapeutics, Foster City, CA, USA 4Donnelly Centre, University of Toronto, Toronto, ON, Canada 5Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 6Department of Biochemistry, University of Toronto, Toronto, ON, Canada *Corresponding author. Tel: +1 416 946 0863; E-mail: [email protected] *Corresponding author. Tel: +1 612 624 6017; E-mail: [email protected] *Corresponding author. Tel: +1 416 978 4939; E-mail: [email protected] EMBO Mol Med (2021)13:e13977https://doi.org/10.15252/emmm.202113977 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to βcatenin-dependent formation of the blood–retina–barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger βcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12−/− mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen-induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction. SYNOPSIS This study reports a FZD4:LRP5 antibody agonist (F4L5.13) that activates βcatenin signaling in endothelial cells. F4L5.13 shows efficacy in animal models by normalizing defective retinal angiogenesis and barrier function, providing a novel therapeutic strategy for eye diseases. βcatenin signaling was activated by F4L5.13, which functions as a Norrin surrogate in endothelial cells. Endothelial barrier function was promoted, and VEGF-induced endothelial permeability was blocked by F4L5.13. Retinal barrier function was restored by F4L5.13 in Tspan12−/− mice. Pathological neovascularization was reduced by F4L5.13 in an OIR model. The paper explained Problem Retinal homeostasis requires an intact blood–retina barrier (BRB) and BRB dysfunction is associated with retinal diseases. In addition, retinal homeostasis requires a proper balance of angiogenesis vs. vascular quiescence. In the retina, the secreted ligand Norrin (NDP) binds FZD4 and the co-receptors LRP5 and TSPAN12 and leads to βcatenin-dependent development of the retinal vascular and BRB formation. Mutations in genes important for this pathway lead to rare congenital eye diseases, such as Norrie disease and FEVR, in which the retinal vasculature and BRB is disrupted, causing impaired vision or blindness. More prevalent diseases such as diabetic retinopathy and macular degeneration are also characterized by neovascularization and blood–retina barrier defects. New therapeutic approaches are needed that actively restore BRB function and normalize aberrant retinal vasculatures. Results We have engineered F4L5.13, a tetravalent antibody, designed to induce FZD4 and LRP5 proximity in order to activate downstream βcatenin signaling. We demonstrated that it is selective for FZD4 and that it efficiently activates βcatenin signaling in mouse brain endothelial cells (bEnd.3 cells) to levels surpassing NDP. F4L5.13 mimics the activity of NDP by activating FZD4 and LRP5 but without requiring TSPAN12. F4L5.13 promotes barrier function in endothelial cells in vitro. In the Tspan12−/− mouse model that exhibits several features of FEVR, systemic injection of F4L5.13 rescued the blood vessel morphogenesis and BRB defects. Finally, when tested in the oxygen-induced retinopathy model, which exhibits hallmarks of neovascular diseases such as retinopathy of prematurity and diabetic retinopathy, the FZD4:LRP5 antibody agonist was found to normalize the observed pathological neovascularization. Impact F4L5.13 is a novel synthetic tetravalent antibody that allows the precise (FZD4 and LRP5 specific) and potent activation of βcatenin signaling in vitro and in vivo. This antibody therefore represents a first-in-class antibody to treat ocular diseases as well as stroke and neurological disorders where endothelial cell barrier function is impaired. Introduction Defects in blood–brain barrier (BBB) and blood–retina barrier (BRB) integrity underlie the ontogeny or progression of a number of diseases such as stroke, pathogen infections, diabetic retinopathy, and neurodegenerative diseases (Obermeier et al, 2013; Sweeney et al, 2019). Wnt/βcatenin signaling is required for CNS angiogenesis and endothelial cell barrier function during development (Liebner et al, 2008; Ye et al, 2009; Wang et al, 2012) and for the maintenance of the BRB and BBB during postnatal tissue homeostasis (Chang et al, 2017; Wang et al, 2018). Among the ten vertebrate Frizzled (FZD) receptors, FZD4 is expressed in endothelial cells where it acts as a cell surface receptor for the secreted proteins Norrin (NDP) (Ye et al, 2009) and WNT7A/B (Cho et al, 2017). Activation of FZD4 leads to βcatenin-mediated regulation of context-dependent genes important for barrier functions, including Sox17, Claudin-5, and MFSD2 (Schäfer et al, 2009; Ye et al, 2009; Wang et al, 2020) and thus limits paracellular permeability and endothelial cell transcytosis (Ben-Zvi et al, 2014). Norrin plays an especially important role for the development and maintenance of the retinal vasculature (Luhmann et al, 2005; Wang et al, 2012, 2018). In this context, LRP5 and TSPAN12 function as co-receptors with FZD4 and are required for Norrin signaling (Junge et al, 2009). Whereas LRP5 and LRP6 are also obligatory co-receptors for Wnt proteins during βcatenin signaling, TSPAN12 acts as a selectivity gate and signal amplifier exclusively in Norrin-induced signaling (Lai et al, 2017). Highlighting the critical roles of Norrin-FZD4 signaling in the retina, NDP, FZD4, LRP5, and TSPAN12 mutations were identified in a number of related congenital diseases with ocular manifestations, such as Norrie disease, osteoporosis-pseudoglioma syndrome, and familial exudative vitreoretinopathy (FEVR; Warden et al, 2007; Baron & Kneissel, 2013; Gilmour, 2015), each of which are associated with hypovascularization of the retina and severe loss of visual function. Overall, disruption of Ndp (Richter et al, 1998), Fzd4 (Xu et al, 2004), Lrp5 (Xia et al, 2010; Chen et al, 2011), or Tspan12 (Junge et al, 2009; Zhang et al, 2018) genes in mice causes similar retinal vascular phenotypes characterized by abnormal blood vessel development and BRB defects. Importantly, endothelial cell-specific expression of a dominant-negative T-cell factor-4 (TCF4) or a stabilized version of βcatenin is sufficient to, respectively, phenocopy or reverse the Ndp and Fzd4 loss-of-function phenotypes, suggesting that Norrin signaling chiefly functions through regulation of a βcatenin endothelial cell transcriptional program (Zhou et al, 2014). Thus, pharmacological activation of FZD4/βcatenin signaling in endothelial cells may represent a novel strategy to promote or restore barrier function by mimicking Norrin and/or WNT7A/B activity and could represent a novel therapeutic opportunity for the treatment of retinal vascular diseases or neurological diseases driven by barrier defects. Results F4L5.13 activates βcatenin signaling in endothelial cells We previously described an induced proximity tetravalent antibody platform called FLAg (Frizzled and LRP5/6 Agonist), which promotes the clustering of Frizzled and LRP5/6 co-receptors and thereby mimics the activity of Wnt proteins to activate βcatenin-mediated transcription (Tao et al, 2019). Given the genetic evidence supporting the role for FZD4 and LRP5 for the development of the retinal vasculature and maintenance of barrier function, we used antibody fragments to assemble F4L5.13, a highly potent and selective FLAg mediating FZD4 and LRP5 activation. F4L5.13 consists of a diabody formed by two identical paratopes recognizing FZD4 fused to the N-terminus of a heterodimeric Fc, and a diabody composed of two distinct paratopes, respectively, recognizing the first two propellers (E1E2; Wnt-1 binding site) and the membrane proximal two propellers (E3E4; Wnt-3 binding site) of LRP5 fused to the C-terminus of the Fc (Fig 1A). F4L5.13 interacts with FZD4 with pM affinity, displays single digit nM binding to LRP5, and importantly, does not interact with any of the 9 other vertebrate FZD receptors and is selective for LRP5 over LRP6 (Fig EV1A–C). Thus, in contrast to pervasive activators of βcatenin signaling such as GSK3β inhibitors or purified Wnt proteins, F4L5.13 should lead to βcatenin-mediated transcriptional regulation in only those cells that co-express FZD4 and LRP5. Figure 1. F4L5.13 treatment activates the Wnt-βcatenin pathway in LEF/TCF reporter cell lines and cultured endothelial cells Top: Molecular architecture of tetravalent F4L5.13. Bottom: Schematic for activation of Wnt/βcatenin signaling by F4L5.13. Activation of βcatenin signaling by F4L5.13 or recombinant NDP (30 nM each) in HEK293T cells transfected with FZD4 and/or LRP5. Values represent fold activation of LEF/TCF reporter gene. Data are presented as mean ± SEM, n = 3. Dose–response curves for the activation of a LEF/TCF reporter gene in HEK293T cells transfected with plasmids encoding FZD4, LRP5 and with either GFP or TSPAN12 by serial dilutions of F4L5.13 or NDP proteins (x-axis). Data are presented as mean ± SEM, n = 3. RT–qPCR of Axin2 expression in bEnd.3 cells treated with serial dilutions of F4L5.13, NDP, or isotype control for 24 h. Data are presented as mean ± SEM, n = 3. RT–qPCR of Axin2 in bEnd.3 cells treated with NDP (200 ng/ml), isotype control or F4L5.13 (1,200 ng/ml) and transfected with control, Tspan12 or Fzd4 siRNAs. Data are presented as mean ± SD, n = 3 technical replicates. Data are representative of two independent experiments. Time course of phosphorylated Dishevelled-3 (p-DVL3) and βcatenin protein levels in bEnd.3 cells treated with 30 nM of F4L5.13 or NDP. Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and βcatenin levels over β-Tubulin measured by densitometry of independent experiments. Data are presented as mean ± SEM, n = 4–6 (*P < 0.05 as compared with NT). Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared with NT). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. F4L5.13 specifically binds FZD4/LRP5 co-receptors F4L5.13 is specific for FZD4, as determined by biolayer interferometry (BLI). F4L5.13 or the isotype control molecule (100 nM) was tested for binding to immobilized recombinant FZD CRDs or the ectodomain of the unrelated receptor Her2. BLI sensors coated with Fc were used to determine the baseline response. Binding kinetics, as determined by BLI, for the binding of immobilized F4L5.13 to FZD4 CRD or LRP5. Experiments were performed in triplicate, and data are presented as the mean ± SD. F4L5.13 selectively binds to LRP5 over LRP6. LRP5/6 proteins coated on a maxisorp plate are recognized in a concentration-responsive manner by F4L5.13 but not by a non-targeting IgG control (4275). F4L5.13 binds to LRP5 with a low nanomolar EC50 value and is > 50-fold selective for LRP5 over LRP6. Data are presented as the mean ± SD from three independent experiments. Activation of βcatenin signaling by WNT3A, F4L5.13, or recombinant NDP (30 nM each) in HEK293T cells transfected with plasmids encoding FZD4 and/or LRP5. Values represent fold activation of LEF/TCF reporter gene. Data are presented as mean ± SEM, n = 3. Binding of F4L5.13 to the cell surface of HEK293T cells overexpressing FZD4 and LRP5 by flow cytometry. Data are representative of two experiments. Download figure Download PowerPoint We first compared F4L5.13 and NDP for their ability to activate βcatenin-signaling using the pBAR reporter, which faithfully monitors LEF/TCF-mediated transcription. Treatment of HEK293T cells (which express only trace amounts of FZD4) with F4L5.13 or NDP required the presence of ectopically expressed FZD4 to detect reporter activation, whereas treatment with WNT3A led to activation in both wild-type cells and when FZD4 was expressed (Figs 1B and EV1D). Supporting these results, flow cytometry experiments detected F4L5.13 binding only in HEK293T cells overexpressing either FZD4 or LRP5 (Fig EV1E). Consistent with the NDP-specific functions described for TSPAN12, co-expression of FZD4 with TSPAN12 led to potentiation of the NDP response but did not affect the activation mediated by F4L5.13 (Fig 1C). We further identified the murine immortalized brain microvasculature cell line bEnd.3 as a highly NDP-responsive cell line. In these cells, F4L5.13 activated expression of the βcatenin target gene Axin2 with similar potency but with higher efficacy than NDP (Fig 1D). Knockdown of Tspan12 led to blunting of the NDP response as previously described (Junge et al, 2009; Lai et al, 2017), but did not affect signaling promoted by F4L5.13 (Figs 1E and EV2A). In contrast, bEnd.3 cells treated with Fzd4 or Lrp5 siRNA were largely unresponsive to either NDP or F4L5.13 (Figs 1E and EV2B–D). Both NDP and F4L5.13 similarly led to Dishevelled phosphorylation and βcatenin stabilization (Fig 1F). We conclude that FZD4 and LRP5 clustering triggered by F4L5.13 is sufficient to activate βcatenin signaling in endothelial cells, and as such, F4L5.13 defines a novel class of FZD4-specific agonists. Click here to expand this figure. Figure EV2. Downregulation of Tspan12, Fzd4, and Lrp5 in bEnd.3 cells A, B. RT–qPCR of Tspan12 (A) and Fzd4 (B) mRNA expression in bEnd.3 cells transfected with scrambled, Fzd4- or Tspan12-targeting siRNA and treated or not with isotype control, F4L5.13 or NDP for 24 h. Data are presented as mean ± SD, n = 2 technical replicates. Data are representative of two independent experiments. C. RT–qPCR of Axin2 in bEnd.3 cells transfected with control or Lrp5-targeting siRNAs and treated or not with isotype control, F4L5.13 or NDP for 24 h. Data are presented as mean ± SD, n = 3 technical replicates. Data are representative of two independent experiments. D. RT–qPCR of Lrp5 mRNA expression in bEnd.3 cells transfected with scrambled or Lrp5 siRNA and treated or not with isotype control, F4L5.13 or NDP for 24 h. Data are presented as mean ± SD, n = 3 technical replicates. Data are representative of two independent experiments. Download figure Download PowerPoint F4L5.13 mimics NDP treatment in bEnd.3 cells We next used RNA-seq analysis to identify differentially expressed genes upon treatment of bEnd.3 cells with F4L5.13 or NDP for 8 or 24 h (Fig 2A, Table EV2). Supporting the results above, F4L5.13 treatment strongly induced Axin2 expression indicating robust activation of βcatenin signaling. Genes differentially expressed following F4L5.13 treatment were enriched for biological processes such as vasculature development/angiogenesis, surface-protein signaling, cell adhesion, and epithelium development (Figs 2B and EV3), consistent with the previously described role of the FZD4-LRP5 signaling axis in CNS vascular development. Importantly, treatment of bEnd.3 cells with NDP for either 8 or 24 h led to differential expression of an overlapping set of genes confirming that F4L5.13 effectively mimics NDP function in endothelial cells (Fig 2A–C). Indeed, following treatment for 24 h, 91% (20/22) of the genes regulated by NDP were also regulated by F4L5.13. A closer examination of expression changes revealed genes linked to GO-Biological Process terms Cell surface receptor signaling pathway (GO:0007166), Cell adhesion (GO:0007155), and Vascular development (GO:0001944) as well as the Wnt signaling pathway (KEGG:04310). When compared with NDP, treatment with F4L5.13 consistently led to increased magnitude of gene induction/repression both by RNA-seq and qPCR validation, which possibly reflects its increased efficacy (Figs 2D and EV4A). Among the genes regulated by both NDP and F4L5.13, Angpt2 was previously shown to be induced by NDP treatment of cultured human retinal microvascular endothelial cells and to mediate, at least in part, the NDP-mediated increase in proliferation (Ohlmann et al, 2010). Another gene strongly induced by both NDP and F4L5.13 was the membrane protein MAL that was previously identified as the receptor for the Clostridium perfringens epsilon toxin (ETX) on endothelial cells and shown to be required for the ETX effects on blood–brain barrier permeability (Linden et al, 2019). Figure 2. F4L5.13 treatment induces expression of vasculature development and cell adhesion programs in cultured endothelial cells mimicking Norrin Volcano plot of gene expression changes in bEnd.3 cells following 24 h of treatment with 30 nM F4L5.13. Genes with significant changes in expression level are highlighted in blue and genes co-regulated by treatment with 30 nM NDP for 24 h are highlighted in red. Statistical analysis was performed using the DESeq2 R package and adjusted P-value (Wald test) threshold less than 0.1 was used. Labeled genes are associated with enriched GO biological processes indicated below. Enrichment Map of overrepresented GO biological processes and pathways in genes regulated by F4L5.13. Node sizes represent the number of genes belonging to individual terms (g:SCS FDR adjusted P-value < 0.05, gProfiler). Venn diagrams showing overlap in differentially expressed genes following treatment with F4L5.13 or NDP. Heat maps of differentially expressed genes mapping to the indicated biological processes and pathways across treatment groups in RNA-seq experiment. Mean of n = 3 samples per group. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Top 20 significant GO biological processes enriched in F4L5.13-treated cells Genes significantly regulated by F4L5.13 treatment were analyzed using the GO biological process annotation, and the top 20 GO terms significantly enriched (FDR adjusted P-value, REVIGO) were shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. RT–qPCR validation of differentially expressed genes identified by RNA-Seq and genes involved in endothelial barrier function in bEnd.3 cells RT–qPCR of a panel of differentially expressed genes identified by RNA-Seq in bEnd.3 cells treated with F4L5.13 or NDP for 8 h and 24 h. Data are presented as mean ± SEM, n = 3–5 biological replicates. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared with NT). RT–qPCR of endothelial junctional genes in bEnd.3 cells treated with F4L5.13 or NDP for 8 h and 24 h. Data are presented as mean ± SEM, n = 3 biological replicates. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test. Download figure Download PowerPoint F4L5.13 treatment promotes endothelial cell barrier function One of the earliest features underlying multiple retinopathies is the Vascular Endothelial Growth Factor (VEGF)-mediated breakdown of the blood–retina barrier and increased endothelial cell permeability. This underlies the current standard of care use of anti-VEGF therapies such as Lucentis (ranibizumab), Eylea (aflibercept), Beovu (brolucizumab), and Avastin (bevacizumab) that revolutionized the clinical management of vascular and exudative diseases of the retina (e.g. macular edema, retinopathy of prematurity, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, and myopic choroidal neovascularization). However, incomplete responses to anti-VEGF therapy and therapeutic resistance are common clinically and lead to persistent disease and significant unmet clinical needs (Gross et al, 2018). Moreover, accumulating evidence indicates that anti-VEGF therapy may not be reversing the nonperfusion underlying pathological diabetic retinopathy (Nicholson et al, 2018). Knowing that FZD4/βcatenin signaling is required for the development and maintenance of CNS endothelial cell barrier properties, we therefore asked whether F4L5.13 treatment could promote barrier functions and oppose the effects of VEGF and determine whether this could represent a novel treatment strategy. Treatment of bEnd.3 cells with F4L5.13 led to increased βcatenin levels and upregulation of ZO-1 expression to comparable levels as NDP (Fig 3A). Then, we investigated the ability of F4L5.13 to rescue endothelial cell barrier breakdown induced by the endothelial cell permeability factor VEGF. When cells were pretreated with VEGF, the endothelial cell barrier was rapidly disrupted, as observed by marked decrease in cell surface localization of tight junction proteins ZO-1, CLDN3, and CLDN5 (Fig 3B). F4L5.13 treatment following VEGF stimulation led to a robust rescue of ZO-1, CLDN3, and CLDN5 cell surface expression indicating that stimulation of FZD4/βcatenin signaling was sufficient to restore tight junction organization disrupted by VEGF treatment (Fig 3B and C). Importantly, in the bEnd.3 cell model βcatenin signaling did not lead to increased Cldn5 mRNA levels, as was previously shown in vivo, or to changes in transcript levels of other cell junction components (Fig EV4B). We conclude that in bEnd.3 cells the effect of F4L5.13 on cell surface levels of junction components is not a result of direct transcriptional regulation. To functionally assess endothelial cell barrier function, we measured the permeability of a confluent endothelial monolayer for 40-kDa FITC-dextran. bEnd.3 cells were grown on transwell filter inserts until confluent and were then treated with VEGF and F4L5.13 simultaneously or were treated with VEGF for 1 h followed by F4L5.13 for 1 h. In both treatment schedules, incubation of bEnd.3 cells with F4L5.13 significantly rescued VEGF-induced permeability (Fig 3D). We conclude from these results that specific activation of the FZD4-LRP5 receptor complex by F4L5.13 promotes cell barrier function, in part, by promoting the assembly of tight junctions. Figure 3. F4L5.13 promotes endothelial cell barrier function Expression of endothelial junction protein ZO-1 and stabilization of βcatenin in bEnd.3 cells treated with 30 nM F4L5.13 or 30 nM recombinant NDP for the indicated times. Blots are representative of five different experiments. Histogram represents the ratio of ZO-1 and βcatenin levels over β-Tubulin measured by densitometry of five independent experiments. Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared with NT). Immunofluorescence of ZO-1, CLDN3, and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1 h. ZO-1 is shown in green, CLDN3/5 in red, and DAPI in blue. Scale bars: 15 µm. Quantification of ZO-1, CLDN3, and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared to VEGF treatment). Transendothelial permeability assay quantifying the passage of FITC-dextran through a monolayer of bEnd.3 cells. Passage of FITC-dextran was measured after exposure of bEnd.3 cells to 100 ng/ml VEGF, 30 nM F4L5.13 or both or pretreated with VEGF for 1 h before treating with F4L5.13 for 1 h. Data are presented as mean ± SD, n = 5 independent experiments. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared to VEGF treatment). Download figure Download PowerPoint F4L5.13 restores retinal vascular d